摘要
羊痒病的检测方法为单克隆抗体介导的免疫学反应，包括ELISA、western blotting和免疫组织化学(IHC)等方法，同时结合脑组织组织病理学观察。本研究通过大肠杆菌融合表达PrP27-30蛋白，制备特异性单克隆抗体，初步寻找单克隆抗体抗原结合位点，对所得单克隆抗体进行免疫学特性分析，选取最佳单克隆抗体，建立我国的羊瘁病免疫组化检测方法，并对我国部分地区采集样品进行普查，在我国羊痒病的监测方面做了比较系统的工作；另外本研究利用突变技术突变单抗2H3结合位点，首次提出并证明羊PrP208位点在不同物种间存在I／M的多态性，这一多态性导致2H3单抗对不同物种PrP亲和力存在差异。
本实验利用PCR方法从小尾寒羊基因组DNA中扩增PrP27-30编码基因，并克隆到硫氧还原蛋白融合表达载体pET32a上，转化至E．coli BL21，IPTG诱导融合表达，得到相对分子质量约为35kD的重组小尾寒羊PrP27-30融合蛋白。以纯化的重组小尾寒羊PrP27-30融合蛋白免疫prp~c基因敲除小鼠，经过两次加强免疫，通过淋巴细胞杂交瘤技术，取脾脏细胞与骨髓瘤细胞进行融合，制备出抗小尾寒羊PrP27-30单克隆抗体，经三次亚克隆，筛选出六株能稳定分泌针对小尾寒羊PrP27-30特异性单克隆抗体的杂交瘤细胞株。
利用大肠杆菌原核表达系统，以相邻肽段间每间隔15个氨基酸(PrP-peptide 2除外)，共6段肽段来分段表达小尾寒羊PrP27-30，分别命名为PrP-peptide 1、PrP-peptide 2、PrP-peptide 3、PrP-peptide 4、PrP-peptide 5和PrP-peptide 6。将6段融合蛋白分别与羊痒病单克隆抗体进行Western blotting反应，根据反应情况分析单克隆抗体抗原结合位点的大概位置，初步得到了待检5株羊痒病单克隆抗体的抗原结合位点，其所在位置分别为：2H3在199aa-213aa之间、4C6在153aa-154aa左右、5F11在154aa-168aa之间、7F1在214aa-227aa之间、7F11在154aa-168aa之间。
对2H3、4C6、5F11和7F11四株单克隆抗体与牛、羊PrP~c的ELISA反应特性，与牛和羊prp~c和prp~(Sc)的Western blotting、免疫组化反应特性等几个方面进行了详细的鉴定和分析。结果显示，2H3对羊PrP~c和PrP~(sc)显示了较强的反应性，对牛Prp~c和PrP~(sc)无反应；4C6对牛和羊PrP~(sc)和PrP~(sc)都有较强反应性；5F11和7F11显示出相似的反应特性，对羊PrP~c和PrP~(sc)反应性较强，而对牛PrP~c和PrP~(sc)次之，抗原结合位点结果与单抗的反应特性相一致。比较清楚的了解了四株单抗的免疫学特性，为每株单抗供不同用途使用提供了理论依据。
根据单抗2H3免疫学特性，利用NCBI中BLAST功能比对单抗结合抗原区段氨基酸的差别，发现羊PrP208位点表现有种属特异性，推测此位点可能影响2H3免疫学特性；利用大肠杆菌原核表达系统表达含有2H3单抗结合区段的PrP肽段，在PCR引物中引入突变，将小尾寒羊PrP208位点的异亮氨酸Ⅰ突变为牛PrP208位点蛋氨酸M，与未引入突变的同一段小尾寒羊PrP肽段一起与2H3进行Western blotting反应，结果显示2H3不与突变后的PrP肽段发生反应，但与未引入突变的同一肽段发生反应。结果说明，2H3的主要抗原结合位点为羊208位点的Ⅰ，此位点的种属差异是造成2H3单抗具有免疫学特性的原因，结果同时也说明2H3在动物朊毒体蛋白研究上具有特殊意义。
在以上研究的基础上，本实验以自制单抗为核心试剂，优化各步骤反应的最佳条件，建立具有我国自主知识产权的羊痒病免疫组化检测方法，并采集我国部分省、市、自治区羊脑样品，利用建立的羊痒病免疫组化检测方法对我国羊痒病进行普查。在2005年检测的3211头份样品和2006年检测的1711头份样品中，所有4922头份检测样品均显示为阴性，初步说明我国目前尚无羊痒病的发生。
The detection of scrapie is authorized by immunological assay mediated by monoclonal antibody, including ELISA, western blotting and immunohistochemistry (IHC) assay, in addition to histopathological test on brain. By the expression of Chinese short-tailed Han sheep PrP27-30 in E. coli BL21 (DE3) and preparation for anti-PrP27-30 of Chinese Short-tailed Han sheep monoclonal antibodies, the binding site and immunological characterization of the mAbs specific to ovine scrapie were systematicly studied in the present research. Two of the suitable mAbs, named 4C6 and 5F11, were selected to establish the method of immunohistochemistry assay. The total of 4922 samples from 26 provinces and autonomous regions of all over China were demonstrated for Scrapie. Meanwhile, using technique of gene mutation on the binding site of mAb 2H3, we discovered that I/M polymorphism of ovine PrP208 could identify ovine prion protein to bovine prion protein.
Prnp gene of Chinese Short-tailed Han sheep was amplified from the Genomic DNA by the polymerase chain reaction and inserted into plasmid pET32a using T4 DNA ligase after digested with restriction enzyme, EcoRⅠand HindⅢ. The recombinant plasmid, named PrP-pET32a, was transferred to E. coli BL21 (DE3). The recombinant Chinese Short-tailed Han sheep PrP27-30 fusion protein, approximately 35kD, was obtained after 4h induced by IPTG. PrP null mice were injected subcutaneously with purified PrP27-30 fusion protein. After two times boosted, SP2/0 cell line and lymphocytes of the immunized PrP null mice were fused by means of lymphocyte hybridoma technique. Six hybridoma cell lines, which stably secreting monoclonal antibodies specific to PrP27-30 of Chinese Short-tailed Han sheep, were selected after three times of subclone.
To find out the binding sites of mAbs 2H3, 4C6, 5Fll, 7F1 and 7F11, overlapping recombinant PrP27-30 which interval 15 amino acids every two consecutive peptides (excluding PrP peptide 2) were expressed in E. coli BL21 (DE3) and named PrP-peptide 1, PrP-peptide 2, PrP-peptide 3, PrP-peptide 4, PrP-peptide 5 and PrP-peptide 6 respectively. Binding sites of scrapie monoclonal antibodies were identified by Western blotting. Binding sites of five monoclonal antibodies specific to ovine scrapie are: 2H3 is between 199aa-213aa, 4C6 is between 153aa-154aa, 5F11 is between 154aa-168aa, 7F1 is between 214aa-227aa and 7F11 is between 154aa-168aa, respectively.
ELISA characterization, western blotting characterization, IHC characterization of PrP~c and PrP~(Sc) between bovine and ovine were identified and analysized by mAbs 2H3, 4C6, 5F11 and 7F11 respectively in present research. The result showed that, 2H3 only reacted with ovine PrP but not bovine PrP; 4C6 strongly reacted with both bovine and ovine PrP; 5F11 and 7F11 gave the similar characterization which reacted more strongly with ovine PrP than bovine PrP.
Acording to immunological characterization of Scrapie mAb 2H3, 2H3 binding site was compared to the other prion protein sequences published in Genebank by BLAST on NCBI web. The result showed that ovine PrP208, Isoleucine (I)/Methionine (M), owned polymorphism. By recombinant DNA technology, mutant fusion protein Mu-PrP-peptide5 which I replaced by M in 208 site was obtained. Western blotting showed that 2H3 only reacted with PrP-peptide5, but not Mu-PrP-peptide5. It implied that I/M polymorphism of ovine PrP208 could identify ovine prion protein to bovine prion protein.
Two of the suitable mAb, 4C6 and 5F11, were selected to establish the method of immunohistochemistry assay. The total of 4922 samples from 26 provinces and autonomous regions of all over China were demonstrated for Scrapie during 2005 to 2006. All of the brain samples were scrapie negative.
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